biocompatibility studies of pectin-fibrin ...2015)/p.55-60.pdfcellulose chemistry and technology...
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CELLULOSE CHEMISTRY AND TECHNOLOGY
Cellulose Chem. Technol., 49 (1), 55-60 (2015)
BIOCOMPATIBILITY STUDIES OF PECTIN-FIBRIN NANOCOMPOSITE
BEARING BALB/C MICE
RESHMI S. NAIR, T. RESHMI, P. RESHMI,* CHANDRAN SARIKA, K. S. SNIMA, UNNI AKK,*
SHANTIKUMAR V. NAIR, K. PAVITHRAN,**
KRISHNAPRASAD CHENNAZHI and
VINOTH-KUMAR LAKSHMANAN
Amrita Centre for Nanosciences and Molecular Medicine, Amrita Institute of Medical Sciences and
Research Centre, Kerala, Kochi-682041, India *Central Animal House Facility, Amrita Institute of Medical Science and Research Centre,
Kerala, Kochi-682041, India **
Oncology Department, Amrita Institute of Medical Science and Research Centre,
Kerala, Kochi-682041, India ✉Corresponding author: Vinoth-Kumar Lakshmanan, [email protected]
Pectin is a natural polysaccharide and the pectin scaffold system has proved to be suitable for an intended use towards
biomedical applications, such as drug delivery and tissue engineering. Studies on gemcitabine loaded pectin-fibrin
scaffold have shown it to be cytotoxic towards ovarian cancer cells at the in vitro level. Our present study aims at
substantiating the biocompatibility of the pectin-fibrin composite scaffold in a mouse implantation model in order to
prove the compatibility of the scaffold system in vivo. Composite scaffolds were implanted and the biocompatibility
was assessed after the 1st, 6
th and 12
th week of study, respectively. Macroscopic inspection of the implantation site
revealed no pathological inflammatory responses and histopathology studies depicted remarkable neutrophil
accumulation within the implant in a timely manner. Furthermore, the immune response indicated significant difference
with cytokines IL-1β, IL-10, and IL-17α, respectively. These results suggested that this scaffold system could be a
promising targeted drug delivery system for the slow release of drugs in a mouse disease model.
Keywords: scaffold, implant, in vivo, macroscopic, histopathology, biocompatible
INTRODUCTION The use of biodegradable nanoparticles as
effective drug delivery devices has shown
significant therapeutic potential.1 It aims to create
new tissues and organs by introducing cells,
biocompatible materials, and supportive factors
and is one of the current applications for the
treatment of various cancer types.2 An optimal
scaffold material would provide both structural
support and act as a reservoir for the release of
bioactive substances.3 This could directly
influence the behavior of colonizing cells, leading
to an advantage in the tissue adaptation. Recent
efforts in this field have highlighted the
importance of drug, protein and growth factor
delivery.4 Numerous biomaterials ranging from
natural to artificial polymers have been
investigated to construct scaffolds for drug
delivery purposes. In order to exploit the
advantages and eliminate the undesired charac-
teristics of individual polymers, various
composite scaffolds comprising two or more
polymers have been considered.2
Pectin is a heteropolysaccharide composed of
1,4-linked-d-galactosyluronic acid residues useful
for the construction of a drug delivery system.
Certain pectins may possess amide groups and in
others the acid groups may either be free,
combined as a methyl ester, or exist as sodium,
potassium, calcium or ammonium salt.5,6
High
and low methyl ester proteins with more than
50% of esterified acid units and less than 50%
methyl ester groups, are the two forms of pectin
extracted from citrus fruits.6 It is an edible
polysaccharide useful for the construction of drug
delivery systems. Pectin functions not only as a
detoxifying agent by helping in regulating and
protecting the gastrointestinal (GI) tract, but also
as invigorating the immune system. Pectin gets
RESHMI S. NAIR et al.
56
digested by the enzyme pectinase, which is
present only in the colon, and therefore remains
undigested in the GI tract, which contains
enzymes like protease and amylase.7 This makes
pectin an ideal drug carrier for colon-specific drug
delivery, which is known to have the advantage of
achieving higher biological availability because
the pH in the colon is neutral and peptidase
activity is relatively lower.8,9
Biodegradable
natural polymers, such as chitin and pectin, have
proven to show good biocompatibility and non-
toxicity.10,11
Fibrin is a fibrous non-globular protein
involved in blood clotting, even a 60-100 mg/mL
of fibrin can initiate coagulation, hampering the
blood flow. Fibrin-chitosan sodium-alginate
composite scaffolds have shown improved
mechanical properties, providing a good
composite for wound dressing applications.12
Another study with chitosan-fibrin-collagen
asymmetric scaffold has shown that fibroblasts
adhered to the walls of the scaffold, suggesting
good growth and excellent cell biocompatibility
of the scaffold.13 Furthermore, in vitro
cytotoxicity studies done with gemcitabine loaded
pectin-fibrin scaffold systems showed more than
70% cell death, proving the cytotoxic behavior of
the drug combination towards ovarian cancer
cells.14 These results suggested the significance of
the scaffold system as an efficient implantable
drug delivery system for the treatment of ovarian
cancer. Here in this work, the biocompatibility of
pectin-fibrin scaffold system is compared with the
implantable pectin scaffold system.
Inflammation is the response of tissue to
injuries, which is categorized into two groups:
responses to acute inflammation and those to
chronic inflammation. This is mediated by a
variety of soluble factors, including a group of
secreted polypeptides known as cytokines. The
process of inflammation is characterized in the
acute phase by increased blood flow and vascular
permeability along with the accumulation of fluid,
leukocytes and cytokines. The development of
cellular and humoral responses to pathogen
describes the chronic phase of inflammation.
These soluble factors are multifunctional.
Cytokines are involved in the extensive networks
that involve synergistic as well as antagonistic
interactions and exhibit both negative and positive
regulatory effects on various target cells in an
autocrine as well as paracrine fashion.15
Further,
in this work, the biocompatibility of the
nanocomposite scaffold is demonstrated by the
studies of histopathology and immune response
analysis, using ELISA assay.
EXPERIMENTAL Materials
Pectin (from citrus) and calcium chloride
(analytical grade) were purchased from Sigma-Aldrich.
A Multi-Analyte ELISArray kit was purchased from
QIAGEN Sciences, Maryland, USA.
Scaffold preparation For the preparation of the pectin scaffold, the
method of ionic gelation was followed.14 Calcium
chloride solution served as ionic cross-linker for the
process of pectin gelation. Chitosan was added to
reduce the hydrophilic nature of pectin, and increase its
stability. 8% w/v of pectin solution was prepared using
double distilled water by constantly stirring at 1200
rpm at 85 °C for 20-25 minutes for obtaining a
monodispersive solution. The solution was allowed to
cool and when the temperature approached 40 °C, 5
mL of chitosan solution (1% w/v) was added to the
pectin solution by constant stirring at 1200 rpm for 15
minutes. The pH was monitored and maintained
neutral. This pectin scaffold served as a control for the
experiment. The composite scaffold of fibrin with
pectin was prepared by redispersing the pectin scaffold
in 2 mL of double distilled water. Further, the solution
was added to 8 mL pectin solution (8%w/v) and kept
under constant stirring speed of 1200 rpm at room
temperature for 15-20 minutes. The remaining steps
for the preparation of the scaffolds were the same as
explained above. Thus, stable nanoscaffolds were
formed. The hydrogels were transferred to 24-well
tissue culture plates and freezed at -20 °C for 24 h. The
frozen samples were then lyophilized (Alpha 2-4 LD
plus Christ, Germany) for 24 h.
In vivo implantation studies
Animal experiments were carried out under a
protocol approved by Institutional Animal Ethics
Committee (Approval no: IAEC/2012/1/5), in six adult
Swiss albino mice, weighing 20-30 g (Figure 1). The
mice were maintained in plastic cages with paddy husk
bedding in a room with ambient temperature and light.
The mice were given sterilized laboratory food and
filtered water.
In the present study, the in vivo evaluation of the
prepared scaffolds was performed. Before
implantation, the scaffolds were cut into sections of 0.7
cm diameter and 0.2 cm thickness. This was incubated
in sterile PBS for 3 days in order to analyze the
porosity of the construct.
Six female Swiss albino mice were selected and the
activities were observed before implantation. The mice
were anesthetized by intramuscular injections of
xylazine and ketamine in the ratio 1:4 (KETMINR 50,
Pectin-fibrin nanocomposite
57
THEMIS Medicare limited) and a 2-cm area of the
dorsal skin was cleaned and shaved. Sub-cutaneous
implants (n = 2) were inserted through a 0.5 cm
incision on the dorsal lower left quadrant and secured
using a PROLENE 4/0 suture stitch. The National
Institute of Health guidelines were followed for both
the care and use of laboratory animals.
Figure 1: Implantation procedure with a 0.7 cm pectin scaffold on the dorsal lower left quadrant
Implant recovery and histology
Mice were sacrificed after periods of 1 week, 6
weeks and 12 weeks, respectively, in groups of 2 (n =
2). At the time of sacrifice, skin tissue, spleen, liver
and sera were removed from each mouse to evaluate
the immune response to the implants. The pectin and
composite scaffold implants were recovered and the
area of implantation was visually inspected for the
evidence of any tissue reaction or inflammation.
Recovered implants were fixed in 10% buffered
formalin, dehydrated, and embedded in the paraffin
blocks. The histological evaluations of the recovered
implants were done using hematoxylin and eosin
staining. Hematoxylin and eosin stain (H&E stain) is a
popular staining method in histology. The staining
method involves application of Hemalum, which is a
complex formed from aluminium ions and hematein,
which is an oxidation product of hematoxylin.
Hemalum gives a blue color to the nuclei of cells (and
a few other objects, such as keratohyalin granules and
calcified material). Nuclear staining is followed by
counterstaining with an aqueous or alcoholic solution
of eosin Y, which colors other eosinophillic structures
with various shades of red, pink and orange.16,17
Immune response studies Serum isolated from blood samples of euthanized
mice was analyzed for immunological reactions. Acute
and chronic inflammatory responses were studied
using a Multi-Analyte ELISArray kit (QIAGEN
Sciences, Maryland, USA). CBA inflammatory kits are
advantageous as they allow the detection of a whole
panel of cytokines in a multiplex fashion, using small
volumes. The Multi-Analyte ELISArray kit is a Mouse
inflammatory cytokine kit designed to simultaneously
profile the level of multiple cytokines or chemokines,
using the conventional and simple sandwich-based
enzyme linked immunosorbant assay (ELISA)
technique.
In our study, the major acute and chronic
inflammatory responses were analysed with the
following cytokines: IL-4, IL-1β, IL-2, IL-10, IL-12,
IL-17α. Acute inflammatory immune responses were
studied using two cytokines, IL-1 β and IL-17 α. IL-1
(α and β) and TNF are extremely potent inducers of
acute inflammation.
RESULTS AND DISCUSSION
Implant recovery and histology analysis
Macroscopic inspection of the implantation
site indicated no pathological inflammatory tissue
responses to the scaffold system. Tissue
overgrowth and sinusoidal congestion of implants
were observed at the later stages. The total
number of cells infiltrating the implant
significantly decreased between week 1 and week
12 in the intra-peritonial (IP). The histological
analysis of the recovered tissue indicated early
neutrophil accumulation within the implant,
which resolved over time. Various tissue sections
were analysed and the following observable
changes were noted (Figure 2).
Considering the spleen, in the first week of
study, we observed that the two sections of the
+control and composite showed widened red pulp
and congestion within the vascular system. The
6th week of study revealed splenic tissue with
RESHMI S. NAIR et al.
58
follicles and areas of haemorrhage and a few
multinucleated cells. After the 12th week,
histology results allowed very similar
observations: widened red pulp with haemorrhage
and giant cells in white pulp.
Skin tissue revealed follicular plugging and
dermis showing neutrophilic infiltrate around the
amorphous substance for the control samples. The
test sample showed skin irregular acanthosis,
follicular plugging and a focus of squamous nests
with keratin pearl formation above dermis with
inflammatory infiltrate. Similarly, after the 6th
week of study, it was observed that the skin tissue
of the control sample presented dermis with an
area of dystrophic calcification and fibrosis, while
the test sample showed increased follicular
plugging within the dermis. After the 12th week,
the scaffolds were found to be degrading;
inflammatory cells with fibrosis were indicative
of neutrophil accumulation. This change was due
to the prolonged implantation of the scaffold
system for a period of 12 weeks, which was
considered to be the major criterion for
confirming the biocompatibility of the scaffold.
In the case of liver tissues, after the 1st week of
implantation, the control tissue samples showed
congestion and ballooning degeneration of a few
cells and mild lymphocytic cells infiltrated around
portal tracts. The test sample showed congested
vessels and sinusoids. After the 6th week, the
control sample exhibited liver tissue with
congested vein and sinusoids, while the test
sample showed mild nucleomegaly in the
hepatocytes. After the 12th week, the sections
showed congested sinusoids and lymphocyte
infiltrate around portal triads. As a result of the
increased sinusoidal congestion and infiltration of
lymphocyte, we could confirm the nature of the
scaffold system over time, by considering the 1st
week and 6th week results.
Figure 2: Histopathology images of the in vivo effects on the spleen, skin and liver tissue samples of control (pectin
scaffold) and test (pectin-fibrin composite) groups, depicting increased rate of infiltration from the 1st week to the 12
th
week (20x)
Immune response analysis The immune response was analysed for the
tissue sections of the mice after the 1st week, 6th
week and 12th week of implantation. The results
revealed an increased reduction in the cytokine
activity of the chronic inflammation linked
interleukins. The following observations were
made: in the case of IL-1β, there was no
significant increase in the temperature of the
animals during the entire course of the study; IL-
1β was found to significantly decrease in the first
and sixth week of study (p < 0.05). IL-1β is
responsible for triggering fever by enhancing
prostaglandin E2 (PGE2).18,19 At the site
inflammation, IL-1β induces release of histamine
from mast cells, while our data reveal significant
reduction when compared to the corresponding
control groups. This was confirmed by the fact
that in the sixth week of study, a significant
difference was observed in the cytokine between
Pectin-fibrin nanocomposite
59
the control and test groups. For IL-17α, the
activation results in the induction of IL-6 activity,
thereby inhibiting TNF production and hence the
acute inflammation response gets reduced.20
In
our study, there is a significant increase in the
first week of study, thereby inhibiting acute
inflammation. For the 6th week, the results
revealed normal levels of IL-17α, while for the
12th week, it showed rapid reduction.
There was no evident change in the levels of
IL-2 and IL-12 cytokines, which correspond to
chronic inflammation. IL-2 was produced in
response to antigenic stimulation, which thereby
increased the suppression of IFN-γ. This indicates
the absence of chronic inflammatory responses in
the control and test group samples. On the other
hand, IL-4 induces CD4+ T cells to differentiate
into TH2 cells, while suppressing the development
of TH1 cells.21,22
It also acts as a B cell, T cell and
mast cell growth factor, it enhances class II MHC
expression on B cells. For the 1st
week, there was
a tremendous decrease in the levels of IL-4, while
after the 6th and 12th weeks, the levels showed
normal levels, indicating the inactivation of the
CD4+ cells, which do not proceed to the T cell
differentiation (Figure 3). IL-10 is the cytokine
synthesis inhibitory factor (CSIF) that inhibits the
IFN-γ production by activated T cells.16
It acts as
anticytokine by inhibiting antigen specific T cell
proliferation. After the 1st week and the 6
th week,
no significant variations of the IL-10 cytokine
from the normal levels were observed. On the
other hand, increased reduction in the levels of
IL-10 after the 12th week indicated the activation
of T cell proliferation in the biological system.
Figure 3: Immune response analysis indicating acute and chronic inflammation with greater response after the 12th
week of implantation in control (pectin scaffold) and test (pectin-fibrin composite) groups
Statistical analysis Statistical analysis of the control and test
groups was carried out and the results are
presented in Figure 3. The values of the immune
response study were expressed as mean ±
standard deviation (SD). To determine the
statistical difference between groups, the two-
tailed Student’s t-test was performed. A
probability value (p) of less than 0.05 was
considered to be statistically significant.
Significant differences were observed for IL-1β,
IL-10, and IL-17α cytokines, respectively.
CONCLUSION Our study elucidated the biocompatibility of a
pectin-fibrin scaffold system, compared with an
implantable pectin scaffold system. The
macroscopic inspection of the implantation site
revealed no pathological inflammatory tissue
responses to the scaffold system. Tissue
overgrowth and sinusoidal congestion of the
implants were observed at the later stages. The
total number of cells infiltrating the implant
significantly decreased between week 1 and week
12 in the intra-peritonial (IP).
RESHMI S. NAIR et al.
60
The histological analysis of the recovered
tissue indicated early neutrophil accumulation
within the implant, which resolved over time.
Various tissue sections were analysed and
marginal inflammatory responses were observed
in the later stage of the study. The results suggest
that this scaffold could be a promising targeted
drug delivery system for the slow release of drugs
in a mouse disease model, confirming the in vitro
and in vivo strategies. The immune response
results of the tissue sections revealed an increased
reduction in the cytokine activity of the chronic
inflammation linked interlukins. Therefore, the
developed scaffold system could be an efficient
implantable drug delivery system for disease
management. In the future, this study can be
extended to test the pectin-fibrin scaffold system
in an ovarian mouse tumor model.
ACKNOWLEDGEMENTS: The authors
acknowledge the Department of Science and
Technology (DST), India, for the partial financial
support by SERB Division (Ref. No.: SR/FT/LS-
147/2009) to Vinoth-Kumar Lakshmanan. Author
K. S. Snima is grateful to the Council of
Scientific and Industrial Research (CSIR), India,
for providing Senior Research Fellowship (09/963
(0030)/2K 13-EMK-I) for carrying out her
research work. Authors Reshmi S., Reshmi T.,
Sarika Chandran thank the Department of Science
and Technology (DST), for MTech grant support.
We are also grateful to Praveen for his help in
nano fabrication. We thank Amrita Centre for
Nanosciences and Molecular Medicine for the
infrastructure support.
REFERENCES 1 S. K. Nitta and K. Numata, Int. J. Mol. Sci., 14,
1629 (2013). 2 P. T. Kumar, C. Ramya, R. Jayakumar, S. V. Nair,
and V. K. Lakshmanan, Colloid. Surf. B Biointerfaces,
106, 109 (2013).
3 P. J. Vande Vord, H. W. Mathew, S. P. DeSilva, L.
Mayton, B. Wu et al., J. Biomed. Mater. Res., 59, 585
(2002). 4 R. R. Chen and D. J. Mooney, Pharm. Res., 20,
1112 (2003). 5 L. Lin Shu, L. F. Marshall, K. Joseph, B. H. Kevin,
Biomaterials, 24, 3333 (2003). 6 M. J. H. Fernandez and J. T. Fell, Int. J. Pharm.,
169, 115 (1998). 7 H. Madziva, K. Kailasapathy, and M. Phillips, J.
Microencapsul., 22, 343 (2005). 8 I. L. Novosel’skaya, N. L. Voropaeva, L. N.
Semenova, S. S. Rashidova, Chem. Nat. Comp. D, 36,
1 (2000). 9 P. Bernabé, C. Peniche, and W. A. Monal, Polym.
Bull., 55, 367 (2005). 10 R. K. Mishra, A. K. Banthia, and A. B. Majeed,
Asian J. Pharm. Clin. Res., 5, 7 (2012). 11 K. M. Zia, M. Zuber, I. A. Batti, M. Barikani, and
M. A. Sheik, Int. J. Biol. Macromol., 44, 23 (2009). 12 M. P. Devi, M. Sekar, M. Chamundeswari, A.
Moorthy, G. Krithiga et al., Bull. Mater. Sci., 35, 1157
(2012). 13 C. M. Han, L. P. Zhang, J. Z. Sun, H. F. Shi, J.
Zhou et al., J. Zhejiang Univ. Sci., 11, 24 (2010). 14 S. Chandran, G. Praveen, K. S. Snima, S. V. Nair,
K. Pavithran et al., Curr. Drug Deliv., 10, 326 (2013). 15 C. A. Feghali and T. M. Wright, Front. Biosci., 2,
12 (1997). 16 J. R. Baker, Microsc. Sci., 103, 493 (1962). 17 G. Avwioro, J. Phys. Chem. Solids, 1, 24 (2001). 18 D. Amsen, K. E. De Visser and T. Town, Methods
Mol. Biol., 511, 107 (2009). 19 P. S. Crosier and S. C. Clark, Semin. Oncol., 19,
349 (1992). 20 R. L. Van Bezooijen, L. Van Der Wee-Pals, S. E.
Papapoulos, C. W. G. M. Lowik, Ann. Rheum. Dis.,
61, 870 (2002). 21 M. P. Beckmann, D. Cosman, W. Fanslow, C. R.
Maliszewski and S. D. Lyman, Chem. Immunol., 51,
107 (1992). 22 T. Yokota, N. Arai, H. D. Vries, H. Spits, J.
Banchereau, et al., Immunol. Rev., 102, 137 (1988). 23 C. D. Nandan, P. Reshmi, S. Uthaman, K. S.
Snima, A. K. K Unni, et al., Nano Pharmaceutics and
Drug Delivery, 1, 30 (2013).